Against the background of an advance in genome-sequence research, attention has been shifted to proteome analysis, in which proteins expressed in living bodies are exhaustively analyzed. Mass spectrometry is a high-sensitivity and high-throughput protein identification method and considered to be one of major approaches for proteome analysis.
Proteome is analyzed by following the procedure described below. First, the molecular weights of peptide fragments resultant from enzyme-catalyzed digestion of protein are measured. Then, the resulting peptide fragments are further dissociated in a mass spectrometer to measure the molecular weights of individual fragments. The molecular weights of original peptide fragments and of their fragments are searched in a database to identify the target protein.
A method for dissociating sample molecules in the mass spectrometer and analyzing the masses of the fragments thereof is called a MS/MS analysis, an essential approach for proteome analysis.
As one of mass spectrometers capable of MS/MS analysis, an ion trap mass spectrometer is well known. (See, for example, Patent document 1, U.S. Pat. No. 2,939,952.) In this ion trap mass spectrometer, RF voltage is applied between a ring electrode and a pair of end cap electrodes composing the ion trap, forming a quadrupole field in the ion trap to trap and store ions. At that time, the introduction of a neutral gas, for example helium gas, causes kinetic energy of ions to be lost because the ions coming into the ion trap collide against the introduced gas, improving efficiency of ion trapping. After being stored, the ions are ejected from the ion trap starting from one having the smallest m/z ratio by scanning the amplitude of RF voltage and detected, forming a mass spectrum (MS spectrum).
MS/MS analysis is performed using an ion trap mass spectrometer by following the procedure described below. First, ions are stored in the ion trap and by following the procedure described above, a mass spectrum is formed. The ion to be dissociated (a precursor ion or parent ion) is selected among those on the resulting mass spectrum. Then, after being stored again in the ion trap, all the ions excluding the parent ion are ejected from there. This step is commonly called isolation.
As one of the parent ion isolation methods, such a method that auxiliary AC voltages are applied to two endcap electrodes. When the amplitude of auxiliary AC voltage exceeds a certain level, the orbits of the ions go into the unstable state, and the ions are ejected from the inner space of the ion trap.
Next, the parent ion remaining in the ion trap is dissociated. Ion dissociation is commonly performed with Collision Induced Dissociation (CID) With CID, auxiliary AC voltage is applied to two end cap electrodes to increase the kinetic energy of the parent ion, causing it to collide and to dissociate it against a neutral gas, for example, helium gas, which is introduced in the ion trap as a target gas. The target gas also serves as a buffer gas for improving ion trapping efficiency.
Since all of or part of the fragment ions resultant from CID remain trapped and stored in the ion trap, finally the mass spectrum of fragment ions (MS/MS spectrum) can be obtained by scanning the RF voltage to eject the fragment ions stored in the ion trap from there starting from one having the smallest m/z ratio and detect them.
With the ion trap mass spectrometer, the MSn (n>2) analysis can be performed, in which the parent ion is further selected among the fragment ions and dissociated into smaller fragments to analyze the masses of them. The MSn analysis provides such an advantage that more detailed information on the structure of the original ion can be obtained. The MSn analysis is performed by following the procedure described below. First, the MS(n−1) analysis is performed and the parent ion is selected among those on the resulting mass spectrum (MS(n−1) spectrum) Next, the steps up to the immediately before the step for obtaining an additional MS(n−1) spectrum are repeated. After ion isolation and dissociation, the mass spectrum (MSn spectrum) of the resultant fragments is obtained.
Such a structure that a quadrupole filter is disposed at the front of the ion trap is known (see, for example, Patent document 2, U.S. Pat. No. 5,572,022). In this structure, ions can be isolated inside the quadrupole filter, enabling ion storage and isolation to be performed simultaneously, which improves the duty ratio for ion trapping and resultantly the detection sensitivity in MS/MS analysis.
Such a mass spectrometer is known that the ion trap and a TOFMS are combined in the direction orthogonal to the direction of ion traveling (see, for example, Patent document 3, JP-A No. 297730/2001). With this type of mass spectrometer, ion storage, ion isolation, and CID are performed at the ion trap and the masses of the ions are analyzed in the TOFMS. Mass analysis is performed by following the procedure described below. After the ions are stored in the ion trap, the application of RF voltage is stopped and an electrostatic field is formed to eject the stored ions. The ejected ions go into the inside of the TOFMS, where is being pumped to a high vacuum. Then the ions are accelerated by an electric field orthogonally to the direction of ion travel and the time-of-flight of the ions are measured.
As mentioned above, in the case of an ion trap mass spectrometer, a neutral gas must have been introduced in the ion trap for two purposes, one being the improvement of ion trapping efficiency and the other being the achievement of CID. The pressure of this neutral gas may affect not only ion trapping efficiency and CID efficiency but also mass resolution of the mass spectrum and isolation resolution.
FIG. 2 is a schematic view explaining the subject of the present invention, which indicates the dependency of the performance (101, 102, 103, 104) of the ion trap mass spectrometer according to a prior art (Patent document 1) on the gas pressure inside the ion trap and the operating gas pressure. In FIG. 2, the horizontal axis indicates the gas pressure inside the ion trap and the vertical axis indicates the levels of the performance (101, 102, 103, 104) (as a value becomes higher, the performance become more enhanced). In FIG. 2, the dependency of CID efficiency 101, ion trapping efficiency 102, mass resolution 103, and isolation resolution 104 on the gas pressure are schematically shown. The dependency of mass resolution 103 and isolation resolution 104 on the gas pressure deteriorate as the gas pressure drops and the gas pressure is attained for providing optimal ion trapping efficiency 102 and CID efficiency 101. On the other hand, no optimal gas pressure is attained for providing all the optimal values of CID efficiency 101, ion trapping efficiency 102, mass resolution 103, and isolation resolution 104. Usually, focusing on ion trapping efficiency 102 and mass resolution 103, the gas-pressure for operating the ion trap is set within the region 105, which provides both of acceptable ion efficiency 102 and acceptable mass resolution 103, as shown in FIG. 2.
The duty ratio of the ion trap of a prior art (Patent document 1) is calculated as follows, considering a typical assumption that 100 ms is required for ion storage, 20 ms for isolation, 30 ms for CID, and 200 ms for mass analysis, respectively;(100 ms)/(100 ms+20 ms+30 ms+200 ms)=0.285.
According to a prior art (Patent document 2), because ion storage and isolation can be simultaneously performed, the duty ratio is calculated as follows:(100 ms)/(100 ms+30 ms+200 ms)=0.303.
In this case, the duty ratio is slightly improved from 0.285 to 0.303. Moreover, since only parent ion is introduced into the ion trap, the injected ions/unit period of time can be reduced and therefore the period of time for storing ions until the ion trap is filled with ions can be increased. As the result, the duty ratio and the detection sensitivity can be improved.
For example, if the period of time for storing ions until the ion trap is filled with ions can be prolonged to 500 ms, the duty ratio will be improved to the value obtained from the formula below:(500 ms)/(500 ms+30 ms+200 ms)=0.684.
For this reason, it is expected that the sensitivity can be improved by a factor obtained from the formula below;0.684/0.285=2.4.
From the descriptions above, it can be known that the main cause for deterioration in duty ratio in the ion trap mass spectrometer is a relatively long dead-time, about 200 ms, during mass analysis.
According to a prior art (Patent document 2), however, the dependency of mass resolution, CID efficiency, and ion trapping efficiency on the gas pressure are identical to those for the ion trap mass spectrometer disclosed in Patent document 1 and no gas pressure cannot be attained for providing all of acceptable performances. For this reason, the gas pressure is set within the same region as that of the ion trap mass spectrometer according to the prior art (Patent document 1).
In the system according to the prior art (Patent document 3), the subject of improving the duty ratio described in Patent document 2 has been spontaneously solved without a quadrupole filter disposed at the front of the ion trap, thanks for high speed of TOF mass spectrometry. This means that even if 100 ms is required for ion storage, 20 ms for ion isolation, and 30 ms for CID, respectively, only less than 1 ms is needed for mass analysis in the TOF system. For this reason, since the duty ratio has already reached to the level obtained from the formula below: (100 ms)/(100 ms+20 ms+30 ms+1 ms)=0.662, without a quadrupole filter disposed. Even if the duty ratio is increased toward a value 1 by omitting the isolation time or prolonging the ion storage period of time as the result of disposing a quadrupole filter at the front of the ion trap, the effect of the sensitivity improvement is small against the new problems of more complicated instrument and increased cost, which occur by the disposition of the quadrupole filter. Consequently, in the system according to the prior art (Patent document 3), no quadruple filter needs to be disposed at the front of the ion trap TOF analyzer only from the knowledge of the improvement of the duty ratio of the system according to the prior art (Patent document 2).
On the other hand, the mass resolution of the TOFMS becomes higher as the initial ion state, namely ion dispersion in the space and energy distribution at the moment of voltage being applied to form a electric field for ion acceleration, are smaller in the direction of accelerating ions. The ion dispersion in the space and the energy distribution are smaller as the gas pressure becomes higher inside the ion trap. That is because since the ion dispersion in the space and the energy distribution are smaller as the gas pressure inside the gas trap becomes higher, the ion dispersion in the space and the energy distribution can be easily controlled in the direction orthogonal to the ion ejected from the ion trap. Thus, the mass spectrometer according to the prior art (Patent document 3) has a feature that higher mass resolution is attained as the gas pressure inside the ion trap becomes higher, contrary to the ion trap mass spectrometer.
FIG. 3 is a schematic view further explaining the subject of the present invention, which indicates the dependency of the performances of the mass spectrometer combining the ion trap and the TOFMS together according to the prior art (Patent document 3) on the gas pressure and the operating gas pressure. In FIG. 3, the horizontal axis indicates the gas pressure inside the ion trap and the vertical axis indicates the levels of the performances (a higher value indicates a higher performance). In FIG. 3, the dependency of CID efficiency 101, ion trapping efficiency 102, mass resolution 103, and isolation resolution 104 on the gas pressure are schematically shown. As known from FIG. 3, the gas-pressure region is attained for providing approximately maximum ion trapping efficiency 102, mass resolution 103, and CID efficiency 101 simultaneously. As shown in FIG. 3, the gas-pressure region 105 for providing all of acceptable ion efficiency 102, mass resolution 103, CID efficiency 101, and isolation resolution 104 is achieved for operating the ion trap.
However, like the ion trap mass spectrometer, the isolation resolution 104 deteriorates as the gas pressure becomes higher. For this reason, the system according to the prior art disclosed in Patent document 3 has a problem that no gas pressure can be attained for providing all of optimal isolation resolution 104, ion trapping efficiency 102, mass resolution 103, and CID efficiency 101.
As described above, with the ion trap mass spectrometer, a neutral gas, for example helium gas, must have been introduced into the ion trap serving as both of a target gas for CID and a buffer gas for improving ion trapping efficiency. Either of CID efficiency and ion trapping efficiency depends on the gas pressure and has optimal values.
On the other hand, mass resolution and isolation resolution deteriorate as the gas pressure becomes higher. For this reason, no gas pressure can be attained for providing all of approximately maximum ion trapping efficiency, mass resolution, isolation resolution, and CID efficiency simultaneously.
In the system according to the prior art (Patent document 2), isolation resolution does not depend on the gas pressure inside the ion trap because a quadrupole filter is disposed at the front of the ion trap for isolating ions there. No gas pressure, however, can be attained for providing all of approximately maximum ion trapping efficiency, mass resolution, and CID efficiency simultaneously.
The mass spectrometer according to the prior art combining the ion trap and the TOFMS together (Patent document 3) has a feature contrary to the instruments according to the prior arts (Patent document 1) and (Patent document 2) in that mass resolution is more improved as the gas pressure becomes higher. Nevertheless, no gas pressure can be attained for providing all of maximum ion trapping efficiency, mass resolution, and isolation resolution simultaneously.